Antibodies are proteins which exhibit binding specificity to a specific antigen. Native antibodies are usually heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy and light chain has at one end a variable domain (VH and VL) followed by a number of constant domains. The variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three complementarity determining regions (CDRs) both in the light chain and the heavy chain variable domains. The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md).
The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and in humans several of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The human IgG isotypes, IgG1, IgG2, IgG3, and IgG4 elicit differential responses due to their sequence differences, which result in differential binding the to Fcγ receptors (Daeron (1997) Annu. Rev. Immunol. 15:203-234) and/or the initial complement component, C1q (Cooper (1985) Adv. Immunol. 37:151). Of the various human immunoglobulin classes, only human IgG1, IgG2, IgG3 and IgM are known to activate complement; and human IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4.
Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment, whose name reflects its ability to crystallize readily. The crystal structure of the human IgG Fc region has been determined (Deisenhofer (1981) Biochemistry 20:2361-2370). In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys 226. The Fc region is central to the effector functions of antibodies.
The effector functions mediated by the antibody Fc region can be divided into two categories, namely effector functions that operate after the binding of antibody to an antigen (these functions involve the participation of the complement cascade or Fc receptor (FcR)-bearing cells); and effector functions that operate independently of antigen binding (these functions confer persistence in the circulation and the ability to be transferred across cellular barriers by transcytosis) (Ward and Ghetie (1995) Therapeutic Immunology 2:77-94).
The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) (see Daeron (1997) Annu. Rev. Immunol. 15:203-234; Ward and Ghetie (1995) supra; Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492).
Several antibody effector functions are mediated by Fc receptors (FcRs), which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcγR, IgE for FcεR, etc. Three subclasses of FcγR have been identified, FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). It is known, for example, that IgG1 and IgG3 isotypes bind FcγRI with a Kd of approximately 109 M−1 (Canfield and Morrison (1991) J. Exp. Med. 173:1483-1491), but that the IgG4 isotype binds approximately 10-fold less affinity, and IgG2 isotype has an affinity for FcγRI more than 1000-fold greater (e.g., at least 106 M1; Canfield and Morrison (1991) supra). Another type of Fc receptor is the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and is composed of an α-chain noncovalently bound to β2-microglobulin. FcRn binds IgGs, internalizes them into endocytic vesicles, and then, through a pH-dependent step, recycles the antibodies back into the serum (Ghetie and Ward (2000) Annu. Rev. Immunol. 18:739-766). This process effectively increases the half-life of IgGs and gives them their nominal half-lives in serum of about 20 days (Ghetie and Ward (2000) supra).
C1q and two serine proteases, C1r and C1s, form the complex C1, the first component of the complement-dependent cytotoxicity (CDC) pathway. To activate the complement cascade, it is necessary for C1q to bind to at least two molecules of IgG1, IgG2, or IgG3, but only one molecule of IgM, attached to the antigenic target (Ward and Ghetie (1995) supra). Based upon the results of chemical modifications and crystallographic studies, the binding site for the complement subcomponent C1q on IgG has been suggested to involve the last two (C-terminal) β-strands of the CH2 domain (Burton, et al. (1980) Nature 288:338-344), with amino acid residues 318 to 337 involved in complement fixation (Burton, et al. (1985) Mol. Immunol. 22(3):161-206).
Key residues of the various IgG isotypes involved in binding to FcRs and C1q have been suggested. The binding site on human and murine antibodies for FcγR have been mapped to the lower hinge region composed of residues 233-239 (numbering as in Kabat, et al. (1991) supra; Woof, et al. (1986) Mol. Immunol. 23:319-330; Duncan, et al. (1988) Nature 332:563; Canfield and Morrison (1991) J. Exp. Med. 173:1483-1491; Chappel, et al. (1991) Proc. Natl. Acad. Sci USA 88:9036-9040). Of residues 233-239, P238 and S239 have been cited as possibly being involved in binding. Other previously cited areas possibly involved in binding to FcγR include G316-K338 (human IgG) for human FcγRI (Woof, et al. (1986) supra); K274-R301 (human IgG1) for human FcγRIII (based on peptides) (Sarmay, et al. (1984) Mol. Immunol. 21:43-51); Y407-R416 (human IgG) for human FcγRIII (based on peptides) (Gergely, et al. (1984) Biochem. Soc. Trans. 12:739-743); as well as N297 and E318 (murine IgG2b) for murine FcγRII (Lund, et al. (1992) Mol. Immunol. 29:53-59). Peptide analysis of IgG1 demonstrates significant binding of residues 256-271 to FcγRIIb (Medgyesi, et al. (2004) Eur. J. Immunol. 34:1127-1135).
Armour, et al. ((1999) Eur. J. Immunol. 29:2613) further suggest the role of residues 230-236 in FcγRI binding. IgG2-like lower hinge regions and mutations at residues G327, S330, and S331 were inactive in binding FcγR as well as in complement-mediated cell lysis. Additional analysis of an IgG2 mutant with point mutations A230S and P231S, indicated reduced binding to the 131H polymorphism of FcγRIIa as compared to IgG2 and approximately equal binding to the 131R polymorphism of FcγRIIa as compared to IgG2. Moreover, this IgG2 mutant favored binding to the FcγRIIb inhibitory receptor. See also, WO 99/58572. A hybrid IgG2/IgG4 antibody, composed of IgG2 CH1 and hinge region fused to IgG4 at about residue P238, has also been disclosed which lacks binding to U937 cells which possess FcγRI (WO 97/11971).
Duncan and Winter (Nature 332:738-40 (1988)), using site-directed mutagenesis, reported that Glu318, Lys320 and Lys322 form the binding site for the murine C1q. The key role of these residues was further suggested by Thommesen, et al. ((2000) Mol. Immunol. 37:995-1004) and in U.S. Pat. Nos. 5,648,260 and 5,624,821. However, it was subsequently shown that these residues are not the same residues in human antibody binding to C1q. Alanine substitutions at positions D270A, K322A, P329A, and P331G of IgG1 have also been demonstrated to significantly reduce C1q binding and complement activation (Idusogie, et al. (2000) J. Immunol. 164:4178-4184).
Residue Pro331 has been implicated in C1q binding by analysis of the ability of human IgG subclasses to carry out complement-mediated cell lysis. Domain swapping between IgG2 and IgG3, as well as IgG1 and IgG4 has been used to dissect the function of residues 292-340, with mutations A330S and P331S eliminating C1q binding and mutation P331S reducing binding to FcγRI (Tao, et al. (1991) J. Exp. Med. 173:1025-1028; Canfield and Morrison (1991) supra; Greenwood, et al. (1993) Eur. J. Immunol. 23:1098-1104). The significance of A330 and P331 was also disclosed by Shields, et al. ((2001) J. Biol. Chem. 276:6591-6604) and in U.S. Pat. No. 6,737,056 and U.S. patent application Ser. No. 11/194,989. Site-directed mutation of P331S in IgG1 (Xu, et al. (1994) J. Biol. Chem. 269:3469-3474) and S331P in IgG4 (Brekke, et al. (1994) Eur. J. Immunol. 24:2542-2547) further suggested the key role of this residue in C1q binding and complement activation. Other reports suggest that human IgG1 residues Leu235, and Gly237, located in the lower hinge region, play a critical role in complement fixation and activation (Xu, et al. (1993) J. Immunol. 150:152A). Amino acid residues 231 to 238 have also been suggested to be necessary for C1q and FcR binding of human IgG1 (WO 94/29351).
Therapeutic antibodies for the treatment of a variety of diseases are known in the art. It is desirable that these antibodies do not provoke an immune reaction toward cells harboring the target antigen. Therefore, there is a need in the art for the generation of therapeutic human or humanized monoclonal antibodies that do not possess any effector functionality, yet retain the typical pharmacookinetics of an IgG. The present invention meets this need in the art.